O型口蹄疫病毒VP1基因原核表达及蛋白纯化

以O型FMDV重组质粒pMD18T-O-VP1为模板，利用PCR技术扩增得到O型FMDV-VP1基因片段，将此基因片段与原核表达载体pET32a连接构建重组表达载体pEq32a-O-VP1，经PCR和测序鉴定后，用WIG诱导归1基因的表达，收集不同诱导时间的菌液，进行SDS-PAGE电泳，摸索掌握最佳诱导时间，切取最佳诱导时间电泳胶片做Westem-blotting，分析检验表达产物与其抗血清的反应性。结果显示，分子量约为45ku的蛋白条带反应显著，能被口蹄疫阳性血清识别，表明FMDV-VP1基因在大肠杆菌中得到高效表达，纯化复性的表达蛋白有望开发为诊断抗原和多肽疫苗。

Prokaryotic Expression and Protein Purification of O-type Foot-and-mouth Disease Virus VP1 Gene

With O-FMDV recombinant plasmid pMD18T-O-VP1 as a template,O-FMDV-VP1 gene fragments were obtained by using PCR amplification.This gene fragments wwas connected with prokaryotic expression vector pET32a to construct recombinant expression vector pET32a-O-VP1.After PCR identification and sequencing,IPTG was used to induce the expression of VP1 gene.Different induction time of the bacterium were collected for SDS-PAGE electrophoresis.The best induction time was mastered.And the electrophoresis film with the best induction time was cut to do Western-blotting.The expression product testing with its anti-serum responsiveness was analyzed.The results showed that the protein with the molecular weight of about 45 ku had significant strip reaction and it could be identified by positive serum of food-and-mouth disease.These results showed that the FMDV-VP1 genes were efficiently expressed in E.coli.The purified expression protein is expected to develop diagnostic antigen and peptide vaccines.